Sequential Injury of the Rabbit Abdominal Aorta Induces Intramural Coagulation and Luminal Narrowing Independent of Intimal Mass

Extrinsic Pathway Inhibition Eliminates Luminal Narrowing

From the Department of Pathology, University of Washington (D.W.C., S.M.S.), and ZymoGenetics (C.E.H.), Seattle, Wash.

Correspondence to Dr Charles E. Hart, ZymoGenetics, 1201 Eastlake Ave East, Seattle, WA 98102. E-mail HARTC{at}zgi.com

	Abstract

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Abstract—We hypothesized that activation of the coagulation cascade is involved in arterial remodeling in response to sequential injury. An active site–inhibited recombinant human factor VIIa (FVIIai) was used to inhibit tissue factor, the primary cofactor in the extrinsic pathway of coagulation, in a sequential balloon injury model of the rabbit abdominal aorta. Single balloon injury produced limited intimal thickening at 3 weeks (intimal area, 0.40±0.05 mm²) and no loss in luminal area (12.2±0.9 mm² before injury and 12.1±0.9 mm² at 6 weeks after injury). Sequential balloon injury, 3 weeks after the first balloon denudation, produced a progressive loss of lumen, with 22% and 47% loss of luminal area, respectively, at 3 and 6 weeks. Luminal loss could not be accounted for by intimal growth (at 3 weeks after sequential injury, the intimal area was 0.47±0.08 mm², <4% of the initial luminal area). Sequential injury acutely produced extensive mural and intramural fibrin deposition. Treatment with FVIIai inhibited both the fibrin deposition and the chronic loss of lumen. At 3 weeks after sequential injury, luminal cross-sectional areas were 9.8±0.6 mm² for control rabbits and 14.3±1.4 mm² for FVIIai-treated rabbits. Neither neointimal area nor cell proliferation was reduced by FVIIai treatment. The intimal cell proliferation index 3 days after injury was 7.6±1.1% in control rabbits versus 5.8±1.1% in treated rabbits (P>0.05). These results indicate that tissue factor is an important mediator of coagulation in repeat injury and implicate the extrinsic coagulation cascade in a blood vessel remodeling response that is independent of neointimal growth but leads to extensive loss of lumen.

Key Words: factor VII • tissue factor • fibrin • arteries • tunica intima

	Introduction

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Pathological stenosis, ie, the inappropriate loss of luminal area, is generally seen, along with plaque rupture, as the key feature as atherosclerotic lesions progress to clinical significance. Until recently, most investigators had assumed that stenosis was the result of encroachment by an increase in lesion mass.¹ This view has been challenged recently, especially in the case of postangioplasty restenosis. In 1993, O'Brien et al² found no proliferative cells in a majority of the primary and restenotic coronary atherectomy specimens tested and suggested that the contribution of neointimal growth to loss of lumen must be small. This hypothesis has been supported by intravascular ultrasound studies that have shown that 60% of luminal loss in postangioplasty restenosis of coronary arteries cannot be attributed to gain in intimal area.^{3 4} The failure of the conventional intimal hypothesis to completely account for loss of lumen led cardiologists to propose that a large part of stenosis development was due to remodeling. The term remodeling suggests that loss of lumen is due to redistribution of the vessel wall and implicates a failure of the normal mechanisms that allows vessels to accommodate wall growth.^{5 6 7 8 9}

The term remodeling has been used to describe different processes in the literature. For the present study, we have adopted the definition of Mulvaney,⁵ who has defined eutrophic remodeling as the process of structural change in a blood vessel with no net growth in the wall constituents. Eutrophic remodeling can occur as either inward remodeling (loss of lumen) or outward remodeling (gain in luminal area); however, the basic tenet is that the relaxed vessel has undergone a structural rearrangement that does not involve either gain or loss of vessel wall mass. By this definition, the more general term, remodeling, refers to any structural changes in a vessel that alter the lumen of a relaxed vessel when measured under standard intravascular pressure.

The eutrophic remodeling concept may be even more important in atherosclerotic progression than in restenosis. In 1987, Glagov et al¹⁰ provided evidence that coronary arteries adapt to large gains in intimal mass by increasing their external circumference, a process termed compensatory enlargement. This adaptation preserves luminal area until a critical limit of intimal mass, representing 40% of preadapted luminal area, is reached. The concept of compensatory enlargement has been confirmed in necropsy studies of both primate and human coronary vessels¹¹ as well as in a case study using sequential intravascular ultrasound.¹² Compensatory enlargement of iliac arteries has also been observed from 2 to 4 weeks after angioplasty in a primate model of atherosclerosis.¹³ Stenosis may therefore represent a failure in compensatory enlargement. Thus, we not only lack a complete explanation for the loss of lumen after angioplasty, but we also have no satisfactory hypothesis for the progressive stenosis that occurs even in atherosclerotic vessels in humans.

Animal studies described as models of restenosis also illustrate the importance of distinguishing neointimal hyperplasia, in response to various procedures, from luminal narrowing. A key study by Langille and O'Donnell¹⁴ showed the role of the endothelium in flow-dependent remodeling of blood vessels to smaller luminal diameters. In this model, denudation injury, a method commonly used to produce intimal hyperplasia, eliminated luminal loss in response to decreased blood flow. Recently, several small animal models have shown that intimal growth can only partially account for loss of gain after angioplasty.^{15 16 17} It is probably correct at this point to say that although intimal mass, especially atherosclerotic mass, may contribute to loss of lumen, loss of luminal area must also depend on loss of the intrinsic ability to accommodate an as-yet-unidentified pathological process that promotes vascular narrowing in injured vessels.

The present study attempts to explore one possible mechanism for pathological narrowing after injury. We hypothesized that luminal loss is at least partially dependent on the procoagulant property of the neointima,^{18 19} a property also described in advanced atherosclerotic lesions.²⁰ In the rabbit, previous studies have shown that single injuries produce little evidence of fibrin formation, yet sequential injuries on a preformed neointima produce extensive mural fibrin deposition.^{21 22 23} We have therefore chosen sequential injury to the rabbit abdominal aorta combined with the use of a specific tissue factor inhibitor (FVIIai) to examine the role of the extrinsic pathway of coagulation in blood vessel remodeling. In order to characterize the remodeling process, we have directly measured both luminal loss, which we have quantified by vascular casting, and intimal hyperplasia.

	Materials and Methods

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Experimental Design
A total of 80 New Zealand White rabbits (3.5 kg) were used. To limit variability, female rabbits were used exclusively. The rabbits were divided into two groups: group 1 rabbits received a single balloon denudation injury; group 2 rabbits received a second balloon denudation 3 weeks after the first (Figure 1). For each group, rabbits were divided randomly into two subgroups. One subgroup received FVIIai, which binds specifically to tissue factor and inhibits activation of the extrinsic pathway of coagulation; the second subgroup served as vehicle control.

View larger version (24K): [in this window] [in a new window]   Figure 1. Rabbits were divided into two groups. In group 1, the effect of a single balloon injury was examined. Group 2 was designed to examine the role of a sequential injury produced 3 weeks after the first balloon injury to the aorta. Subgroups of rabbits were treated with FVIIai (i) or vehicle (v). FVIIai was administered by high-dose intravenous bolus injections given preoperatively and postoperatively, followed by low-dose intraperitoneal infusion for 1 week. For group 2 rabbits, FVIIai was administered only at the time of the second injury. In all cases, n represents the number of rabbits in each group.

Rabbits in each group were killed either at 4 hours (n=4), 72 hours (n=6), or 3 weeks (n=6) after the final injury. For the 3-week time point, rabbits were perfusion-cast to provide accurate luminal areas. For all prior time points, the vessels were perfusion-fixed. The 4-hour time points were used to examine mural and intramural fibrin deposition by scanning electron microscopy and immunohistochemistry. For the 72-hour time point, rabbits were infused with BrdU before they were killed for study, and the tissue was used to examine cell proliferation.

To determine whether the luminal loss was progressive, 7 more rabbits were cast 6 weeks after a second balloon denudation injury, and 4 more rabbits were cast at 6 weeks after a single injury. To obtain baseline values, the vessels from 5 rabbits (age- and weight-matched to the 6-week time point) were cast without any vascular injury. An additional 8 rabbits (4 FVIIai-treated and 4 vehicle control rabbits) were killed at 4 hours and 10 days after sequential injury in order to obtain fresh frozen tissue for immunohistochemical study.

Balloon Denudation
Rabbits were anesthetized with intramuscular and intravenous injections of ketamine (Ketaject, Phoenix) and xylazine (Rompun, Miles Inc). A 4F Fogarty embolectomy catheter (Baxter) was inserted via a left femoral arteriotomy and passed 20 cm into the abdominal aorta. The catheter was inflated and drawn back to the iliac bifurcation. Catheter insertion and inflation were repeated for three passes, after which the catheter was removed, the femoral artery was tied, and the incision was closed. The analgesic buprenorphine (0.06 mg, Buprenex, Reckitt and Coleman) and the antibiotic penicillin G (60 000 U, Phoenix) were applied intramuscularly.

For group 2 rabbits, a second balloon denudation was performed 3 weeks after the first procedure. The procedure was identical except that the right femoral artery was used for access and a 3F Fogarty balloon used for denudation. The flow reduction produced by occlusion of both femoral arteries was measured in two rabbits. A transonic flow probe (Transonic Systems Inc) was placed around the central abdominal aorta, and mean flow was repeatedly recorded before and after clamping of the femoral arteries. Clamping produced only minor changes in flow rate, with a mean flow reduction of 9%.

Plasma samples (in 10% sodium citrate, Sigma Chemical Co) were collected at three time points: (1) before balloon denudation (and before administration of FVIIai or vehicle), (2) immediately after ballooning, and (3) 3 days after the final balloon denudation. For rabbits surviving >3 days, a final plasma sample was drawn before they were killed for study. Plasma samples were stored at -80°C for later analysis.

Drug Preparation and Administration
FVIIai was generated from recombinant wild-type human FVIIa as previously described.²⁴ FVIIai produced a dose-dependent prolongation of coagulation times in a standard partial thromboplastin coagulation assay with normal rabbit plasma and rabbit thromboplastin.

The FVIIai dosage was based on the short half-life (30 minutes in rabbits) and a requirement for high circulating levels of FVIIai during the perioperative period. Intravenous bolus injections of FVIIai (6 mg, in Tris-buffered saline, pH 7.4) were administered 30 minutes before balloon denudation, immediately after catheter insertion, and 2, 4, and 8 hours after denudation. For prolonged administration, two osmotic minipumps (model 2mL1, Alzet) were loaded with 2 mL of FVIIai (3 mg/mL) and implanted intraperitoneally. These pumps supplied FVIIai at 60 µg/h over the 1-week period following injury. Control rabbits were given bolus doses of vehicle (Tris-buffered saline, pH 7.4) and were implanted with two osmotic minipumps loaded with vehicle. Animals in the sequential injury group were given the same drug regimen at the time of the second injury.

For cell replication studies, BrdU (Sigma) was administered intraperitoneally in a loading dose (40 mg in 2 mL H₂O) and continuously for 24 hours with an osmotic minipump (model 2001, Alzet) delivering 1.1 mg of BrdU/h. Rabbits were killed 24 hours after initial administration.

Tissue Preparation
Rabbits were heparinized (1000 IU IV, Elkins-Sinn) and killed by anesthetic overdose. The thoracic aorta was cannulated, and the arterial system was flushed with 120 mL of lactated Ringer's solution. For the 4-hour and 3-day time points, the aortas were perfusion-fixed for 20 minutes at 100 mm Hg with 3% paraformaldehyde (phosphate-buffered, pH 7.4; time and pressure were optimized to attain adequate fixation while limiting extensive use of fixative). The abdominal aorta from the renal to the iliac bifurcation was resected and fixed overnight in 3% phosphate-buffered paraformaldehyde. To obtain luminal morphometry, aortas at the 3- and 6-week time points and the uninjured control aortas were cast with Batson's compound (Batson's No. 17, Polysciences) infused at an infusion pressure of 120 mm Hg, which was maintained until the compound had set. The aorta and cast were resected and fixed ex vivo for 1 week in 3% phosphate-buffered paraformaldehyde.

Three portions of each abdominal aorta (1 cm in length) were taken between lumbar vessels beginning with the intralumbar region 2 cm from the iliac bifurcation and progressing proximally (see Figure 2). For aortic casts, the 1-cm segments were cut from the cast with a hand drill with a carbide blade. Portions of the aorta were then gently pulled from the cast. The cast was preserved for determination of luminal diameter and area, and tissue segments were paraffin-embedded. For scanning electron microscopy, sections of aorta were postfixed in 1% glutaraldehyde (phosphate-buffered, pH 7.4), pinned open longitudinally, fixed in osmium tetroxide, critical point–dried, and sputter-coated.

View larger version (97K): [in this window] [in a new window]   Figure 2. To obtain accurate luminal geometry, the vasculature was cast under a transluminal pressure of 120 mm Hg. A representative cast of the vasculature from an uninjured rabbit is depicted. The arrows indicate the three regions, proximal (P), central (C), and distal (D), at which the abdominal aorta was sampled to measure cross-sectional areas and take tissue sections.

Histomorphometry
Cross sections of abdominal aorta were stained with the following: (1) Verhoeff's elastic tissue stain to differentiate the elastic lamellae, (2) hematoxylin and eosin, and (3) Gomori's trichrome connective tissue stain.¹⁸ Because of the large size of the aortic cross sections, a single low-power digitization proved to be less reproducible than a sampling technique in which cross-sectional areas were determined from multiple fields of x200 magnification. For each Verhoeff's stained cross section, eight randomly chosen fields were digitized. With this sampling technique, 50% of the entire cross-sectional area was measured. Optimas image analysis software (Bioscan Inc) was used to measure intimal and medial areas as well as the linear portion of the circumference for each sample field. The latter measurement was divided into the area calculation for each field, and the mean intimal area– and medial area–to–field length ratios were calculated. The vessel circumference was measured under low power, and this measurement was multiplied by the area–to–field length ratio to calculate the total cross-sectional area for each vessel cross section. For measurement of EEL, area low-power magnification was used to digitize the perimeter length of the EEL. Because of possible distortion of the lumen in tissue processing, a cylindrical assumption was used to calculate areas from perimeter length.

For each abdominal aorta, sections from the proximal, central, and distal regions were analyzed. The mean values for each animal were used in the statistical analysis.

Immunohistochemistry and Fluorescent Staining
Immunohistochemistry was performed with the avidin biotin complex system (Vectastain, Vector Laboratories). Paraffin sections were cleared, rehydrated, and washed in PBS (pH 7.4). A 10-minute pepsin (Biomeda) digest at 37°C followed by a PBS wash was performed; the sections were then blocked with 2% horse serum. Sections were incubated with primary antibody for 1 hour at 37°C, washed, and incubated with a biotinylated secondary antibody for 1 hour at 37°C. After the sections were washed, the streptavidin–horseradish peroxidase conjugate was applied, and the sections were stained with diaminobenzidine. For each run, a negative control was processed by deleting the primary antibody and applying an isotype-specific control antibody (Zymed).

Frozen sections were thawed, washed in PBS, dehydrated in ethanol, and acetone-fixed for 10 minutes at 4°C. Endogenous peroxide was blocked with a 5-minute incubation with 1 part hydrogen peroxide (30%) in 9 parts methanol. Sections were then rehydrated to PBS. Tissue blocking and antibody staining proceeded as described for paraffin sections.

The primary monoclonal antibodies used were specific to smooth muscle -actin (Sigma), fibrin (No. 350, American Diagnostica Inc), and BrdU-labeled DNA (Biomeda). Before BrdU staining, DNA was denatured by application of 2N HCl for 20 minutes. To characterize the fibrin-specific antibody, rabbit plasma was clotted with bovine thrombin and embedded in gelatin. Citrated plasma was also embedded in gelatin. Frozen sections were then fixed in 3% phosphate-buffered paraformaldehyde and stained. The antibody was demonstrated to be plasma clot specific in the rabbit.

FVIIai was conjugated with the fluorescent dye Oregon Green 514 by use of a labeling kit (Molecular Probes). FVIIai was dialyzed to PBS (pH 7.4), and 200 µL of 2 mg/mL FVIIai was reacted with 20 µL sodium bicarbonate (pH 8.3) and 7.8 µL dye solution (10 mg/mL in dimethyl sulfoxide) for 90 minutes at room temperature. The reaction was terminated by the addition of excess hydroxylamine, and labeled FVIIai (OG-FVIIai) was purified with a spin column.

For OG-FVIIai staining, paraffin sections were cleared, rehydrated, and washed in Tris-buffered saline (pH 7.4). OG-FVIIai was diluted 1:20 in Tris-buffered saline containing 20 mmol/L CaCl₂ and applied to the sections for 20 minutes. The sections were then washed for 10 minutes with three changes of Tris-buffered saline. To control for specificity, 20x excess unconjugated FVIIai was used to compete off OG-FVIIai. All sections were mounted with Vectashield (Vector Laboratories), and micrographs were immediately taken on an Olympus BH-2 fluorescent microscope.

In Vitro Plasma Assays
In order to measure FVIIai serum levels, a monoclonal-polyclonal sandwich ELISA was used. An anti-human factor VIIa monoclonal antibody (courtesy of Walt Kisiel, University of New Mexico, Albuquerque) was plated onto 96-well microtiter dishes (Maxisorp, Nunc) by adding a concentration of 2.0 µg/mL and a volume of 100 µL to each well, and the plates were incubated overnight at 4°C. The plates were then washed in washing buffer (PBS and 0.05% Tween 20, pH 7.4) and subsequently blocked for 2 hours at 37°C with blocking buffer (PBS, 0.05% Tween 20, and 1% BSA, pH 7.4). Dilutions of FVIIai (20 to 0.027 ng/mL) or sample plasma were made in blocking buffer, 100 µL of test sample or plasma was added to the plates, and the plates were incubated for 1 hour at 37°C and subsequently washed. A rabbit anti-human FVIIa polyclonal antibody (courtesy of W. Kisiel), diluted 1:1000 in blocking buffer, was added to the wells, and the plates were incubated for 1 hour at 37°C. Goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma) was then added, and the plates were incubated for 1 hour at 37°C. The plates were subsequently washed, and the substrate (O-phenylenediamine dihydrochloride) was added. The absorbance was read at 490 , and FVIIai concentrations were determined from a standard curve.

Plasma was also examined for prolongation of partial thromboplastin time in a standard assay with rabbit thromboplastin (Sigma). Duplicate cuvettes containing 50 µL of rabbit plasma and 150 µL of Tris-buffered saline (10 mmol/L Tris and 0.15 mol/L NaCl) were loaded into an automatic coagulation timer (Electra 800, Medical Laboratory Automation Inc). Recalcification was accomplished with a 1:3 dilution of thromboplastin in Tris-buffered saline with 25 mmol/L CaCl₂, and time to clot was recorded. Plasma levels of FVIIa were determined for control samples with a modified coagulation time assay specific for FVIIa, as described by Wildgoose et al.²⁵

Statistical Analysis
Statview software (Abacus Concepts Inc) was used for statistical analysis. Comparisons of drug-treated groups with control groups at each time point were made with the Student t test. An ANOVA and Fisher protected least significant differences were used to compare the effect of single and double injury at the various time points. Paired t tests with a Bonferroni correction were used to compare individual plasma samples over the time course of the experiment. All values in the text are quoted as mean±SEM.

	Results

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Single Injury
The frequency of BrdU-positive cells within the media was elevated 72 hours after a single balloon injury. In vehicle-treated animals, total proliferating cells per cross section were 204±16, with all proliferation confined to the media (7.9% proliferation index) (Table 1). At 3 weeks after the first injury, intimal thickening was observed in all animals. Mean intimal and medial cross-sectional areas were 0.402±0.05 and 1.17±0.09 mm², respectively. Intimal thickening 3 weeks after a single injury represented only 3% of the total luminal area as measured from cast cross sections (luminal area, 13.38±0.5 mm²). Mean luminal areas did not change significantly after a single injury at either 3 or 6 weeks after ballooning (12.3±1.0 mm² for uninjured control animals and 13.38±0.5 mm² and 12.2±1.0 mm² for 3- and 6-week time points, respectively) (Figure 3).

View larger version (16K): [in this window] [in a new window]   Figure 3. Luminal areas for each vehicle control group as measured from abdominal aortic casts are represented. Only the groups subjected to sequential injuries exhibited a significant decrease in luminal area compared with the uninjured control group (*P=0.03, **P<0.001). For sequential injuries, reduction in luminal area was progressive with time.

Treatment with FVIIai had no statistically significant effect on the level of medial cell replication 3 days after injury (200±21 BrdU-labeled cells per cross section, Table 1). Similarly, FVIIai treatment had no effect on intimal growth or on luminal area at 3 weeks after balloon injury (Figure 4).

View larger version (24K): [in this window] [in a new window]   Figure 4. Cross-sectional areas measured from aortas cast 3 weeks after a single or sequential injury (6 weeks from the first injury). For a single injury, treatment with FVIIai had no statistically significant impact on intimal, medial, or luminal area. In sequential injuries, FVIIai treatment eliminated the luminal loss associated with the vehicle control group. However, intimal and medial cross-sectional areas were not statistically different with FVIIai treatment (open bars, vehicle control group; solid bars, FVIIai-treated group). *P=0.03 vs control group. A post hoc analysis revealed no significant difference between neointimal area after single or sequential injury (P=0.48).

Sequential Injury
At 4 hours after sequential injury, histological cross sections displayed an intact neointima. The mean number of BrdU-positive cells (per cross section) in the media 72 hours after the second injury was low (33±20, representing a proliferation index of 0.79%), with cell proliferation largely confined to the preformed intima with a mean of 178±40 cells labeled (7.6% proliferation index) in the intima of vessel cross sections. Total cell proliferation per vessel cross section was similar to the single injury when both intimal and medial cells were considered (211±60 for sequential injury versus 204±32 for a single injury). Proliferation indices in the intima 72 hours after the sequential injury were similar to indices in the media after a single injury (see Table 1).

Sequential injury produced a progressive loss of luminal area not seen in animals injured only once (Figure 3). At 3 weeks after sequential injury, the mean luminal area, as determined from direct measurements of aortic casts, was 80% of that from vessels of uninjured animals (12.3±0.5 mm² [uninjured] versus 9.8±0.6 mm² [injured], P=0.03). At 6 weeks after the second injury, mean luminal diameter had dropped to 55% of the uninjured vessel diameter (12.3±0.5 mm² [uninjured] versus 6.8±0.5 mm² [uninjured], P<0.001).

FVIIai treatment eliminated the luminal narrowing observed at 3 weeks after the second injury (mean luminal cross-sectional areas from casts were 14.3±1.4 mm² for FVIIai-treated vessels versus 9.8±0.6 mm² for control vessels, P=0.03 by t test) (Figure 4). The greatest loss in luminal area in control vessels was seen in the distal abdominal aorta (2 cm proximal to the iliac bifurcation), and the effect of FVIIai treatment was greatest in this location (data not shown). Measurements of EEL area from histological cross sections were also made for analysis of total vessel area. The EEL measurements, 13.6±1.2 and 10.0±0.7 mm² for FVIIai-treated and control vessels, respectively, also showed an increased total vessel area in FVIIai-treated animals (P=0.03) similar to the direct luminal measurements made from casts.

Luminal narrowing could not be attributed to gain in intimal area because there was no difference between intimal area in control and treated vessels (Figure 4). FVIIai treatment also had no significant effect on cell replication rates, as measured by BrdU staining after 3 days (Table 1). Vessel wall structure examined by light microscopy revealed no morphologically distinct differences between FVIIai-treated and untreated vessels.

Fibrin Deposition After Single and Sequential Injuries
No fibrin deposition was observed on aortic cross sections taken 4 or 72 hours after a single balloon injury, and immunohistochemistry with a fibrin-specific antibody displayed only background staining (data not shown). However, in control treated animals receiving sequential injury, extensive fibrin deposition was observed 4 hours after injury. One third of the vehicle control rabbits had mural thrombus visible on gross inspection. All sequential injuries had morphologically visible fibrin on the luminal surface, as evidenced by light microscopy of Gomori's stained sections and scanning electron microscopy (Figure 5a). In sequential injury, the extent of fibrin deposition on the luminal surface was variable, ranging from scattered deposits to extensive coverage of the injured arterial surface. Immunohistochemical staining for fibrin revealed extensive mural and intramural fibrin deposition in control aortas examined 4 hours after a second injury. Dense fibrin staining was observed in the preformed intima (Figure 6a). This dense staining did not extend past the internal elastic lamina; however, scattered regions of staining for fibrin did appear in both the media and adventitia of control vessels. At 10 days after the second injury, fibrin staining was still observed in the intima, with regions of intense staining near the luminal surface and the internal elastic lamina (Figure 6c).

View larger version (173K): [in this window] [in a new window]   Figure 5. Scanning electron microscopy of the luminal surface 4 hours after sequential balloon denudation. Extensive fibrin deposition was observed in vehicle control rabbits (a, x4000 original magnification); in contrast, fibrin deposition was not observed in FVIIai-treated rabbits (b, x6600 original magnification). Platelets were observed on the vessel wall of both treated and control rabbits. Bar=1 µm.

View larger version (116K): [in this window] [in a new window]   Figure 6. Immunohistochemical staining for fibrin at 4 hours after sequential injury revealed extensive deposition within the neointima (i) with some lighter staining within the media (m) of vehicle-treated rabbits (a, x1000 original magnification; iel indicates the internal elastic lamina). FVIIai treatment virtually eliminated intramural fibrin deposition (b, x400 original magnification). At 10 days after sequential injury, a significant amount of fibrin staining remained in the intima of vehicle control rabbits (c, x200 original magnification). Fibrin staining was not observed in vessels from FVIIai-treated rabbits taken 10 days after sequential injury (d, x200 original magnification).

Mural and intramural fibrin deposition was largely eliminated in FVIIai-treated animals examined 4 hours after the second balloon injury. In order to assess the extent of fibrin deposition within the vessel wall and on the luminal surface, three distinct methods were used. First, extensive areas of the vessels were examined for mural thrombus with Gomori's stained sections. The extent of mural fibrin deposition was assessed semiquantitatively in a double-blind manner and scored from 0 to 4. Sections from rabbits receiving vehicle scored significantly higher (mean score of 3) than did sections from FVIIai-treated rabbits (mean score of 0). Second, the surface of vessels 4 hours after sequential injury were examined by scanning electron microscopy. Fibrin and platelet-rich thrombi were frequently seen in scattered regions of the luminal surface of vessels from rabbits treated with vehicle (Figure 5a). In FVIIai-treated vessels, fibrin was not observed on the luminal surface of specimens examined by scanning electron microscopy (Figure 5b). However, limited platelet deposition was observed on the luminal surface of these vessels. Third, the extent of intramural fibrin deposition was assessed by immunohistochemical staining of aortic cross sections with a fibrin-specific antibody. FVIIai-treated vessels displayed very limited staining within the vessel wall at both 4 hours and 10 days after the sequential injury (Figure 6b and 6d). In contrast, large dense patches of staining were observed within the intima of vessels from vehicle control rabbits.

Localization of Tissue Factor
To examine the spacial localization of tissue factor within the vessel wall, tissue cross sections were stained with fluorescent FVIIai (OG-FVIIai). Uninjured vessels displayed intense staining only in the adventitia (not shown). At 3 days after a single injury, diffuse staining developed throughout the media and developing intima (Figure 7a). Three weeks after a single balloon denudation injury, OG-FVIIai displayed intense staining in the neointima and diffuse staining in the media (Figure 7b). At 4 hours after sequential injury, intense staining in the intima could still be observed in vessels of vehicle control rabbits (Figure 7c). The intense intimal staining with OG-FVIIai occurred in the same regions as the antifibrin staining after sequential injury in control treated animals (Figure 6a). Rabbits treated with FVIIai displayed very little OG-FVIIai staining (Figure 7d), indicating that the tissue factor in these sections was not available for binding. OG-FVIIai staining could also be eliminated by coincubation with 10-fold excess unlabeled FVIIai (not shown).

View larger version (114K): [in this window] [in a new window]   Figure 7. Probing tissue sections with fluorescently labeled FVIIai produced diffuse staining throughout the media and developing intima of vessels 3 days after a single injury (a, x100 original magnification). By 3 weeks after one injury, extensive staining was localized in the neointima, indicating an accumulation of tissue factor in the lesion (b, x400 original magnification). At 4 hours after sequential injury, the neointima of control animals maintained a high level of reaction with OG-FVIIai (c, x400 original magnification). FVIIai-treated rabbits, however, displayed only background staining at 4 hours after sequential injury (d, x100 original magnification), indicating that tissue factor in these vessels was not able to react with OG-FVIIa.

Plasma Coagulation Factors
Intravenous injections of FVIIai at the time of the operation produced high circulating levels immediately after the operation, as measured by ELISA on plasma samples (Table 2). Lower circulating levels were maintained for 1 week after the operation by intraperitoneal delivery from osmotic pumps. Consistent with the ELISA results, plasma partial thromboplastin clotting times in the immediate postoperative period were significantly elevated. However, by 3 days after the operation, despite the low levels of circulating FVIIai, partial thromboplastin times were slightly depressed in both treated and control animals. There were no statistical differences for plasma levels of FVIIai or for partial thromboplastin times in rabbits in the single or sequential injury group. Values for the sequential injury group are summarized in Table 2.

	Discussion

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We believe that this is the first report of a mechanism controlling loss of luminal area after arterial injury that is independent of changes in intimal cross-sectional area. We found that sequential balloon injury to the rabbit abdominal aorta produced a progressive loss of luminal area (22% loss at 3 weeks and 47% loss at 6 weeks after injury). Unlike other animal models, this structural rearrangement was associated with negligible gain in vessel wall area. After sequential injury, the neointimal cross-sectional area did not significantly increase, and at 3 weeks after injury, it represented only 4% of the luminal area. In contrast, Gerdes et al²⁶ have shown that 2 weeks after a single injury to the rat or rabbit carotid artery, the neointima is 75% and 30%, respectively, of the luminal area. Because of the large loss of luminal area compared with the negligible vessel wall growth in our model, we have defined the process as inward eutrophic remodeling. The most striking finding after sequential injury was extensive fibrin deposition both on the vessel surface and within the vessel wall. Fibrin deposition and loss of lumen was inhibited by systemic administration of FVIIai, a competitive inhibitor of the extrinsic pathway of coagulation. These results imply that the extrinsic pathway of coagulation is a major mediator of eutrophic remodeling in the rabbit.

Activation of the coagulation cascade has been implicated in both smooth muscle cell mitogenesis and chemotaxis^{27 28 29 30} ; however, in terms of neointimal formation, results have not been consistent between animal models. Short-term application of the direct thrombin inhibitor, hirudin, decreases neointimal area after injury of the rabbit carotid yet has no effect in the rat or minipig.²⁶ Jang et al²⁴ demonstrated that both FVIIai and tissue factor pathway inhibitor decreased the extent of stenosis and intimal area (measured at stenotic regions) after sequential injury to the iliac artery in hypercholesteremic rabbits, whereas hirudin and tick anticoagulant peptide had no significant effect. Our studies using a sequential injury to the aorta in normocholesterolemic rabbits showed that inhibition of the extrinsic pathway with FVIIai had no significant impact on either neointimal area or smooth muscle cell proliferation (measured 3 days after injury), indicating that coagulation is not the major stimulus for proliferation in this model. These results are consistent with the findings of Ragosta et al,³¹ who have shown in a balloon angioplasty model in the hypercholesterolemic rabbit that hirudin treatment was not associated with decreased early smooth muscle cell proliferation despite a significant reduction in plaque size.

As with any animal model, the results from the present study may not be directly predictive of remodeling in diseased human coronary arteries. Possible limitations of the model described herein involve the use of the rabbit abdominal aorta, an elastic vessel that is structurally distinct, with no attempt to develop focal stenoses or plaques. Rather, we have concentrated on the remodeling of the entire vessel in response to sequential mechanical injury. In rabbit models involving hypercholesteremia, focal intimal lesions that are rich in inflammatory infiltrates develop. Remodeling in these hypercholesteremic vessels is clearly multifactorial and involves extensive intimal thickening. The strength of the model that we describe in the present study is that pathological eutrophic remodeling to smaller luminal diameters can occur in vessels with no extensive intimal thickening or inflammation, indicating a distinct mechanism of arterial remodeling. We believe, but have not proven, that this distinct eutrophic remodeling process is one component of vessel remodeling that can occur along with other remodeling processes (such as intimal growth) in mechanically injured severely atherosclerotic arteries.

In the model described in the present study, loss of lumen was not driven by an enlarging neointima. However, there was a weak inverse correlation between luminal area and intimal area in the untreated group (r²=0.412, P=0.04 for correlation). This correlation suggests that properties of the neointima are involved in the inward eutrophic remodeling process. One possibility is that a larger neointima produces more extensive fibrin deposition and that the fibrin is important for the eutrophic remodeling response. There are several possible mechanisms by which coagulation may be involved. The fibrin that we observed could lead to increased and chronic platelet deposition. Platelets have been implicated in the vessel response to injury hypothesis and may participate in vessel remodeling by release of a number of potent biological factors that can stimulate smooth muscle cell replication, migration, matrix production, and contraction of fibrin or collagen gels.^{1 32 33 34 35 36 37 38} Platelet deposition has previously been linked to fibrin deposition in sequential injuries. Groves et al¹⁸ found an 50% reduction in platelet deposition when rabbits receiving a sequential injury were treated with heparin. In the present study, we cannot accept or reject the role of platelets in vessel narrowing. Although platelets were seen reacting with the luminal surface in both FVIIai-treated and vehicle-treated vessels (Figure 5), the study was not designed to quantify the platelet interaction with the vessel over the entire postoperative period. Harker et al,³⁹ however, have demonstrated that FVIIai treatment significantly reduces platelet-rich thrombus deposition at sites of carotid endarterectomy in the baboon.

An alternative hypothesis is that luminal narrowing may involve clot retraction. Retraction in the usual sense as mediated by stimulation of platelets in a platelet-rich clot seems unlikely because of the prolonged time course of vessel narrowing. Our observation of fibrin in the vessel wall, however, raises the possibility that deposition of fibrin within the neointima may act as a scaffolding for cell migration and wound contraction. Fibrin deposition within the vessel wall may also lead to collagen synthesis and scar contracture. Although we found no large differences in histological structure of the collagen fibers, the study was not designed to assess changes in collagen structure or synthesis. Fibrin degradation products have been shown to be mitogenic for smooth muscle cells.⁴⁰ Deposition of fibrin within the extracellular matrix as we have described provides increased complexity, since eutrophic remodeling may involve multiple interactions with structural proteins, resident smooth muscle cells, fibrin/fibrinogen, fibrin degradation products, and lytic enzymes.

Fibrin deposition within the vessel wall may have relevance to human restenosis. The core of the atheromatous plaque has been demonstrated to be extremely thrombogenic,⁴¹ and fibrin, fibrin degradation products, and cross-linked fibrinogen have all been found in human atherosclerotic plaques.^{42 43 44 45 46 47 48} Elevated plasma fibrinogen levels are associated with increased risk of both atherosclerosis^{49 50} and postangioplasty restenosis,⁵¹ and increases in circulating fibrinolytic parameters have been associated with atherosclerotic progression.^{52 53} Although the precise role of fibrin in plaque pathogenesis is yet to be determined, the balance between fibrin deposition and lysis may be intimately involved in vessel remodeling.

The role of coagulation in postangioplasty restenosis remains unclear. Inhibitors of thrombin (recombinant hirudin and hirulog) have not produced either clinical or angiographic improvement.^{54 55} However, the risk of bleeding complications with these compounds limits the extent of anticoagulation that can be achieved within the tissue. Because of their tissue specificity, inhibitors of tissue factor may produce more favorable results.

Our observation of extensive fibrin deposition after reinjury is consistent with previous evidence of an increase in the ability of the rabbit neointima to stimulate coagulation only when reinjured.^{18 21 22 23 56} Groves et al¹⁸ demonstrated the propensity for a sequential injury, performed 1 week after the first balloon denudation to the rabbit aorta, to stimulate mural deposition of platelet-fibrin thrombi. Our studies suggest that the procoagulant state persists within the neointima for at least 3 weeks after the initial injury and that at this time the neointima promotes extensive intramural fibrin deposition. Accumulation of tissue factor within the neointima, as demonstrated by the OG-FVIIai staining, suggests a causative mechanism for this procoagulant response (Figure 7).

Other studies have demonstrated increased tissue factor–FVIIa complex activity within the artery wall after balloon injury.^{19 57 58} In the rat, tissue factor activity is elevated in explants from the carotid media at 1 to 2 hours after balloon injury and returns to near basal levels within 24 hours.¹⁹ Tissue factor activity has been demonstrated in the subendothelium of rabbit aortic explants,^{59 60 61} and luminal tissue factor activity has been shown to be elevated for 16 hours after a single balloon injury to the rabbit abdominal aorta.⁶² Our studies show that tissue factor protein levels in the neointima remain elevated for at least 3 weeks after a single balloon injury to the rabbit abdominal aorta. These results suggest that tissue factor accumulates and persists within the neointima but does not promote fibrin deposition until the vessel is reinjured.

The functional activity of tissue factor in vivo is controlled by a number of factors. Transport and partitioning of coagulation factors, flow-dependent enzyme kinetics, and anionic phospholipid concentrations can all play important roles.^{63 64} It is likely that sequential injury in our rabbit model produced cell disruption and increased permeability, which effectively altered the microenvironment within the neointima to favor coagulation. The high bolus doses of FVIIai that we applied in the acute postoperative period inhibited fibrin deposition and, by inference, tissue factor activity. Bolus doses also prolonged the partial thromboplastin times, whereas the lower maintenance doses (delivered by osmotic pump) did not. Although inhibition of systemic coagulation may not have been attained at later periods, it is possible that the low levels of circulating FVIIai did have a significant impact on maintaining inhibition of tissue factor within the vessel wall.

In summary, we have developed a model of sequential arterial injury that produces chronic inward eutrophic remodeling. The rabbit model described in the present study allows for analysis of the extrinsic pathway of coagulation on vessel wall response to injury. The first injury in this model induces a hypercoagulable state within the vessel, which may be similar to the atherosclerotic plaque. The second injury can represent interventional procedures used to treat sites of vascular stenosis. Our findings using FVIIai to inhibit tissue factor suggest that activation of the extrinsic pathway of coagulation plays a key role in the eutrophic remodeling response.

	Selected Abbreviations and Acronyms

 

BrdU = bromodeoxyuridine EEL = external elastic lamina FVII = factor VII FVIIa = activated FVII FVIIai = active site–inhibited FVIIa OG-FVIIai = Oregon Green FVIIai

	Acknowledgments

 
Dr Courtman was supported by a fellowship from the Heart and Stroke Foundation of Canada. The authors thank Debra Gilbertson and Gayle Yamamoto for technical assistance and Ula Hedner and Mirella Ezban of NovoNordisk for supplying recombinant FVIIa.

Received September 23, 1997; accepted March 2, 1998.

	References

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